As a nucleator of branched actin filaments, the actin-related protein2/3 (Arp2/3) complex fulfills in a key role in remodeling the actin filament network, a major component of the cellular cytoskeleton. The complex is essential for changes to cell shape associated with specific processes, such as endocytosis in yeast, and is required for the formation of actin-based structures, such as actin patches and lamellipodia. Our proposed research seeks to address the fundamental questions about the mechanistic basis of Arp2/3 activation that remain unanswered due to a lack of crucial biochemical and structural data of the active complex. Our goal is to determine the precise atomic interactions that activate the Arp2/3 complex. Accordingly, using Schizosaccharomyces pombe, the fission yeast, we will employ directed mutations of specific amino acid residues in the Arp2/3 complex to determine how mutations at these sites affect the binding of the complex to the sides of pre-existing (mother) actin filaments and nascent (daughter) actin filaments. We will use mutagenic primers for site-directed mutagenesis, along with homologous recombination, gene knock-out and gene replacement, to create mutant strains. PCR and DNA sequencing will be used to genotype mutant strains, while Western blot will reveal expression levels of mutant genes. Mutant complexes will be purified through GST-VCA affinity, ion exchange, and gel filtration chromatography. Phenotype analysis of mutants will be visualized with GFP-tagged markers using confocal microscopy and total internal reflection microscopy (TIRF). We will carry out spectroscopic assays to measure the kinetics of actin polymerization dynamics of mutant Arp2/3 complexes as well as their binding affinity to ATP, actin monomer, the pointed-end of the daughter filament and nucleating-promoting factors (NPFs), such as Wiskott- Aldrich syndrome protein (WASP) family. Additionally, we will use x-ray crystallography to determine the structural basis for Arp2/3 activation by WASP family proteins for some mutants identified in the mother and daughter filament binding experiments that exhibit high affinity for VCA, an activating region of the WASP proteins. The results of our study will offer a detailed model of the interfaces between the Arp2/3 complex and the mother and daughter actin filaments and how WASP proteins bind to the complex to contribute valuable insight to understanding how the Arp2/3 complex is regulated.